The long-term goal of our laboratory is to characterize cis- and trans- acting factors that regulate initiation of DNA replication in mammalian chromosomes. Several origin mapping techniques have been devised which suggest that DNA replication initiates in a 55kbp region between the DHFR and 2BE2121 genes in Chines hamster ovary cells. Preliminary results from our lab show that, as in yeast, a cell cycle-regulated hypersensitive site and a nuclease-resistant domain occur over one of the DHFR locus preferred initiation sites, ori-Beta. As the ORC complex has been implicated in playing a casual role in formation of these chromatin structures, and as these chromatin structures have been implicated in a regulatory role in initiation of DNA replication, two hypotheses have been generated: (i) the organization of chromatin at ori-Beta in CHO cells is involved in origin function, and (ii) the chromatin structures are caused by locally-bound ORC complex(es). These hypothesis will be addressed by the following Specific Aims: 1) Determine whether the chromatin modifications correlate with DHFR origin activity in vivo. Ligation-mediated PCR techniques will be used to identify regions of chromatin modification in hemizygous CHO cells so that the chromatin state in initiation-positive CHO cells can be compared to the chromatin state in initiation-negative CHO cells. This will demonstrate that the chromatin configuration is (or is not) associated with a functional origin. 2) Determine the distribution/binding sites of ORC complex(es) within the DHFR locus and specifically at ori- Beta. Novel approaches involving cross-linking of DNA-interactive factors will be used to determine where ORC binds within the DHRF origin locus. Experiments will then be performed addressing the association of these ORC:DNA interactions with origin function in vivo. Investigation of these questions will allow for a better understanding of the initiation of mammalian DNA replication and will provide a more clear understanding of the mechanisms regulating the G1/S transition.